In vitro and in vivo antiplasmodial evaluation of sugar-modified nucleoside analogues

Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing antimalarial therapies has complicated the problem, and the emergence of resistance to artemisinin derivatives is even more concerning. It is increasingly difficult to cure malaria patients due to the limited availability of effective antimalarial drugs, resulting in an urgent need for more efficacious and affordable treatments to eradicate this disease. Herein, new nucleoside analogues including morpholino-nucleoside hybrids and thio-substituted nucleoside derivatives were prepared and evaluated for in vitro and in vivo antiparasitic activity that led a few hits especially nucleoside-thiopyranoside conjugates, which are highly effective against Pf3D7 and PfRKL-9 strains in submicromolar concentration. One adenosine derivative and four pyrimidine nucleoside analogues significantly reduced the parasite burden in mouse models infected with Plasmodium berghei ANKA. Importantly, no significant hemolysis and cytotoxicity towards human cell line (RAW) was observed for the hits, suggesting their safety profile. Preliminary research suggested that these thiosugar-nucleoside conjugates could be used to accelerate the antimalarial drug development pipeline and thus deserve further investigation.

Malaria continues to be a global health concern, with 247 million infections and 625,000 deaths worldwide in 2021, mainly among children and pregnant women 1 . The WHO African Region is responsible for a disproportionately large amount of the worldwide malaria burden. Human malaria is caused by five species of the mosquito-borne parasitic protozoa of the genus Plasmodium, the most prevalent and deadly of which is Plasmodium falciparum (Pf), which is accountable for about 90% of malaria-related deaths 2 . Despite various malaria control and eradication attempts, most countries have not been able to eradicate the disease 3 . Drug resistance, toxicity, the lack of an effective vaccination, and low drug efficiency are all factors contributing to this 4 . The ongoing battle against Plasmodium drug resistance involves the exploration and development of wide range of therapeutics 5 . The current vaccine used for prevention of Pf malaria, RTS, S/AS01 6 provides only moderate protection, although a pipeline of new vaccine candidates provides optimism. Further, the effective use of the frontline chemotherapy, Artemisinin Combination Therapy (ACTs), is now threatened by the emergence of resistance 7 .
Due to the lack of a vaccine and the growing resistance to current drugs, it is extremely important to develop new antimalarials and, in particular, to design drug candidates with a different structure and mechanism of action than those of current therapeutics 8 . Because pathogenic protozoa lack the ability to synthesize purines via de novo pathway, they rely on the salvage and reutilization of preformed purines for development and proliferation. They have various enzymes that are not found in mammals and can be used as therapeutic targets 9 . Nucleoside and nucleotide analogues are among the most promising class of potential antimalarial drugs because they can act as inhibitors in both the de novo pathway for pyrimidine nucleotide biosynthesis and the salvage pathway
Biological evaluation. Antimalarial activity by SYBR green I based fluorescence assay. To begin with, cellbased assay was performed to evaluate the inhibitory activities of all 26 compounds (1-26) at 1 µM and 10 µM concentrations on the asynchronous culture of Pf3D7 chloroquine-sensitive strain. Growth of one intraerythrocytic cycle of Pf was monitored after treated with compounds whereas untreated parasites were considered as control. At 10 µM concentration, the majority of the molecules inhibited more than 50% of the parasites (Fig. S1). Five of the twenty-six compounds, morpholino-nucleoside hybrid 1 and thiosubstituted nucleoside analogues 7, 16, 17 and 18 were observed to have significant growth inhibition in a dose dependent manner against Pf3D7. Morpholino-nucleoside hybrids 3 and 5 also showed remarkable actvity against Pf3D7 but they have cytotoxic properties (vide infra). Other compounds, except for the above mentioned seven derivatives showed insignificant percent growth inhibition as compared to untreated control against 3D7 strain of Pf.
Among the configurationally altered, thiosubstituted analogues, the thiosugar-containing derivatives were found to have excellent activity against Pf (Fig. S1). The configuration of the thiosugar affected the antimalarial activity to some extent, compounds having gluco-and galacto-configured substituents (7, 16-18) showed excellent, while the mannopyranoside conjugate 15 showed a more moderate growth inhibitory effect. However, it is important to note that even the mannopyranoside-containing nucleoside 15 showed a stronger inhibitory effect (~ 50% inhibition at 1 μM concentration) than the alkylthio-substituted compounds 10, 11 and 19 (~ 5-25% inhibition at 1 μM), suggesting that the thiopyranoside-nucleoside conjugates can have significant antimalarial potential. We have found that the place of substitution did not affect the antimalarial activity, the introduction of a suitable thiosugar in either the 5'-(7) or 3'-position (16)(17)(18) resulted in highly active derivatives.
Our study with compounds 7-9 suggests that appropriate lipophilicity is crucial for the activity of thiosubstituted nucleoside analogues. While the deacetylation (7 → 8) did not significantly affect the antimalarial activity, Table 1. Thio-click-based synthesis of 3'-modified nucleoside analogues 11-18. a a The reactions were performed in a borosilicate vessel by irradiation with a Hg-lamp (λ max = 365 nm) without any caution to exclude air or moisture. b Diastereomeric ratio of products determined by 1 H NMR. c Overall yield of products isolated by column chromatography. d The reaction was performed at − 40 °C. www.nature.com/scientificreports/ the removal of the lipophilic trityl groups (7 → 9) resulted in a significant decrease in growth inhibitiory effect (Fig. S1A). For the 1-thiosugar-containing hit compounds, the lipophilicity value is clogP ~ 5 ( Table 2). The antimalarial effect of purine nucleoside analogues based on the inhibition of purine metabolism pathway is well documented [10][11][12] , but there are hardly any results in the literature for pyrimidine-type antimalarial agents 24 . In this context, it is noteworthy that among the new derivatives tested in this study, several pyrimidine nucleoside analogues (1, 3 and 4 from the morpholino-nucleoside hybrids and 12, 15-18 and 20 from the thiosugar-nucleoside chemotype) showed good/excellent inhibitory effect against Pf.

Effect of compounds on human cell lines.
Effect of compounds on human RBCs was checked with the help of spectrophotometry by measuring the lysis of human red blood cells (hRBCs). Compounds were tested at 0.5, 1, 5, 10 and 20 μM concentrations with 10% (v/v) RBCs suspension for 1 h and observed the percentage of RBCs lysis by compounds 7, 16, 17, 18 and 1.
No significant lysis of erythrocytes was observed at concentration of 20 µM (Fig. 5A), while compounds 3 and 5 showed toxicity (Fig. S3). To evaluate the toxic effects of the potent compounds on human RAW cells, MTT assay was carried out and no apparent toxicity effect was observed for compounds 7, 16, 17, 18 and 1 up to 500 µM concentration (Fig. 5B). Table 2 summarizes the in vitro bioassay results and clogP values of the hit compounds and the reference compound chloroquine.

Mitochondrial membrane disruption potential (Δψ m ).
Mitochondrial membrane potential is a marker of mitochondria's functional status. Modification in mitochondrial membrane potential leading to mitochondrial dysfunction triggers cell death 25,26 . Mitochondrial dysfunction was detected by using membrane permeant JC-1 dye. JC-1 is a cationic probe, due to electronegative environment inside the functionally active mitochondria with high Δψm, it aggregates in energised mitochondria and gives red color fluorescence at 590 nm, but in case of low Δψm (depolarized state), JC-1 remain in monomeric form and gives green color fluorescence at 530-10 nm. Decreased ratio of red/green fluorescence intensity of compound treated sample as compared to untreated control indicates depolarized mitochondrial membrane 27,28 . The damage to the parasite's mitochondria after treatment with compounds 7, 16, 17, 18 and 1 was measured according to the method reported previously 28 . Because of lipophilic nature of the JC-1, it is cell permeable and emits a green signal (525 nm) in the www.nature.com/scientificreports/ cytoplasm, but the high transmembrane potential of functional mitochondria causes it to aggregate and emit a red signal (590 nm). In contrast, as shown in Fig. 6, the parasite treated with compounds showed a considerable decrease in JC-1 red staining and an increase in diffused green mitochondrial fluorescence. The loss of mitochondrial membrane potential was demonstrated by a significant fall in the red/green ratio of the JC-1 stained counts in parasites treated with compounds. The parasites treated with IC 50 concentrations of compounds 7, 16, 17, 18 and 1 exhibited reduced red/green ratio of 5.26 ± 1.13, 4.98 ± 1.77, 6.28 ± 1.55, 5.53 ± 1.40 and 5.69 ± 0.70. The untreated control parasite showed the red/green ratio of 7.04 ± 1.30 ( Fig. 6A and B).
In vivo antimalarial activity of compound 7 against P. berghei ANKA. The most potent compound, the 1-thioglucose-adenosine conjugate 7 with purity level of > 95% (Fig. S4) was evaluated for antiplasmodial activity in mice model. In vivo antiplasmodial efficacy of 7 was assessed in P. berghei ANKA infected BALB/c mice model. Compound dose was given intra-peritoneal injections for seven days in a row. To check the parasitemia, thin blood smears were made from P. berghei ANKA infected mice for up to seven consecutive days. www.nature.com/scientificreports/ At a dose of 50 mg/kg, compound 7 showed 60% inhibition against the rodent-infecting P. berghei ANKA. The percentage parasitemia of group of mice treated with 7 was observed to be 10% as compared with the control wherein percentage parasitemia was 25% on the seventh day (   www.nature.com/scientificreports/ of mice compared to the control group (Fig. 7B). To make relevant inferences, statistical analyses were carried out on the in vivo data using Graphpad Prism data sheets.

Conclusion
Drug-resistant malaria has emerged as a severe threat to worldwide malaria management, necessitating the development of novel antimalarials that are effective against both drug-susceptible and drug-resistant malaria. Hybrid molecules that utilizes new entities with different pharmacophores represent a rational approach in development of novel therapeutics. The present work demonstrates that nucleoside-1-thiosugar hybrids, as synthetic products without cytotoxicity, might be suitable candidates for the development of antimalarial agents against Pf. The IC 50 concentrations of the aforementioned nucleoside analogues were determined on Pf 3D7 and PfRKL-9 strains and were found to be between 0.95 µM and 2.21 µM. These analogues, when applied at IC 50 concentrations, resulted in depolarization of the membrane potential of treated malaria parasites, leading to cell death. A 50 mg/kg dose of hit compound 7 administered to mice infected with the rodent parasite P. berghei ANKA showed up to a 60% reduction in parasite load, along with prolonged survival of the mice. In addition to 1-thiosugar-nucleoside conjugates, our study also identified morpholino-nucleoside hybrids as a new chemotype of antimalarial drug candidates. Unfortunately, in the investigated morpholino-nucleoside hybrids, the strong antimalarial effect was usually accompanied by significant cytotoxicity, therefore further optimization is required for this chemotype in order to develop effective and safe antimalarial lead compounds.

Materials and methods
General. Nucleoside derivatives 1-6 17 , 10 16 and 19 18 , naphtylmethylmercaptane 27c 29 , Fmoc-cysteine derivative 27d 17 , 1-thiosugars 27f-i 30 , adenosine-4'-exomethylene 28 16 , uridine-2'-exomethylene 29 31 , uridine-3'-exomethylene 30 16 and 5-methyluridine-3'-exomethylene 31 16 were prepared according to literature procedures. . Two-dimensional COSY and 1 H − 13 C HSQC experiments were used to assist NMR assignments 16 . The diastereomeric ratio of the new compounds were determined on the basis of their 1 H NMR spectra as described earlier 18,19 . MALDI-TOF MS measurements were carried out with a Bruker Autoflex Speed mass spectrometer equipped with a time-of-flight (TOF) mass analyzer. In all cases 19 kV (ion source voltage 1) and 16.65 kV (ion source voltage 2) were used. For reflectron mode, 21 kV and 9.55 kV were applied as reflector voltage 1 and reflector voltage 2, respectively. A solid phase laser (355 nm, ≥ 100 μJ/pulse) operating at 500 Hz was applied to produce laser desorption and 3000 shots were summed. 2,5-Dihydroxybenzoic acid (DHB) was used as matrix and F 3 CCOONa as cationising agent in DMF. ESI-QTOF MS measurements were carried out on a maXis II UHR ESI-QTOF MS instrument (Bruker), in positive ionization mode 32 . The following parameters were applied for the electrospray ion source: capillary voltage: 3.5 kV; end plate offset: 500 V; nebulizer pressure: 0.8 bar; dry gas temperature: 200 °C and dry gas flow rate: 4.5 L/min. Constant background correction was applied for each spectrum; the background was recorded before each sample by injecting the blank sample matrix (solvent). Na-formate calibrant was injected after each www.nature.com/scientificreports/ sample, which enabled internal calibration during data evaluation. Mass spectra were recorded by otofControl version 4.1 (build: 3.5, Bruker) and processed by Compass DataAnalysis version 4.4 (build: 200.55.2969). The photoinitiated reactions were carried out in a borosilicate vessel by irradiation with a low-pressure Hglamp (Osram Supratec UV, HTC 150-211, 150 W, 230 V, R7s) giving maximum emission at 365 nm, without any caution to exclude air or moisture 16 . The experimental set-up consists of the reaction vessel and the cooling medium (acetone-liquid nitrogen mixture) in a Dewar flask and an UV-lamp. Before irradiation, the entire set-up is covered by an aluminum foil tent to protect the laboratory personnel against UV light 16,30 . The purity of hit compounds was assessed on HPLC system (Gilson, USA) with an analytical column (C18) and a Thermo Separation Spectra SERIES UV100 detector coupled with software. The mobile phase for the analysis was prepared from acetonitrile and water (v/v) and compounds showed purity > 95%.
The study was carried out accordingly to relevant guidelines. Mice-based in vivo experiments are reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org).
Chemical synthesis. General method for photoinitiated free radical thiol-ene reaction 16,30 . The corresponding alkene, thiol and DPAP (0.1 eqiuv/alkene) were dissolved in the given solvent to obtain a solution with a concentration range of 0.2-0.5 M for the alkene (the solution should be as concentrated as possible). The reaction mixture was cooled to the given temperature and irradiated with UV-light for 15 min. After irradiation another 0.1 eqiuv. of DPAP was added and the irradiation continued for another 15 min. The addition of 0.1 equiv. of DPAP and the irradiation was repeated two more times. The solvent was evaporated in vacuo and the crude product was purified by flash column chromatography.